Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(2)
Immunocytochemical Analysis of Tissue Sections
Small pieces of epididymal tissue were snap frozen in iso-pentane at — 80°C and embedded in Cryo-M-Bed (Bright) medium. Sections 8-12 l^m thick were cut on a cryostat, dried onto clean glass slides, and stored at —20°C until analysis. Dried sections were immersed in PBS containing 3% (w/v) BSA at room temperature for at least 1 h to block nonspecific binding sites, washed 2 X 10 min in PBS, and probed with 2D6 McAb supernatant for 1-16 h at 4°C. Slides were washed 3 X 10 min in PBS followed by incubation with FITC-RAM diluted 1:100 in PBS/1% (w/v) BSA for 3 h in the dark at room temperature.
After washing 3 X 10 min in PBS/0.1% (w/v), BSA sections were mounted in Citifluor antifade (Ci-tifluor, London, UK) and viewed with UV light using a Zeiss Axiophot photomicroscope (Carl Zeiss, Oberkochen, Germany) fitted with epiflu-orescence illumination and a SPOT-RT camera (Diagnostic Instruments, Sterling Heights, MI).
Immuno-Gold Electron Microscopy
Pieces of rat epididymis from the proximal caput, distal caput, and cauda regions were immersed in 3% (w/v) paraformaldehyde in 0.1 M phosphate buffer containing 5% (w/v) sucrose for 2 h at room temperature. Tissues were then transferred to 0.2 M sodium cacodylate buffer (pH 7.2) and cryoprotected by immersion in 2 M sucrose containing 5% (w/v) polyvinylpyrrolidene overnight at 4°C.